Fixation

Incubating the cells in 100% methanol (chilled at -20°C) at room temperature for 5 min.

Using 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperature.

The cells should be washed three times with ice cold PBS.

Antigen retrieval (optional step)

Certain antibodies work best when cells are heated in antigen retrieval buffer. Please check the product datasheets for recommendations for each primary antibody being used.

Preheat the antigen retrieval buffer (100 mM Tris, 5% (w/v) urea, pH 9.5) to 95°C. This can be done by heating the buffer in a coverglass staining jar which is placed in a water bath at 95°C.

Using a small pair of broad-tipped forceps, place the coverslips carefully in the antigen retrieval buffer in the coverglass staining jar, making note of which side of the coverslips the cells are on.

Heat the coverslips at 95°C for 10 min.

Remove the coverslips from the antigen retrieval buffer and immerse them, with the side containing the cells facing up, in PBS, in the 6-well tissue culture plates.

Wash cells in PBS three times for 5 min.

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Permeabilization

If the target protein is expressed intracellularly, it is very important to permeabilize the cells.

Acetone fixed samples do not require permeabilization.

Incubate the samples for 10 min with PBS containing either 0.1 - 0.25% Triton X-100 (or 100 μM digitonin or 0.5% saponin). Triton X-100 is the most popular detergent for improving the penetration of the antibody. However, it is not appropriate for membrane-associated antigens since it destroys membranes.

​The optimal percentage of Triton X-100 should be determined for each protein of interest.

Wash cells in PBS three times for 5 min.

Blocking and incubation

Incubate cells with 1% BSA, 22.52 mg/ml glycine in PBST (PBS+ 0.1% Tween 20) - for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 10% serum from the species from which the secondary antibody was raised in. Please see antibody datasheet for recommended blocking).

Incubate cells in the diluted antibody in 1% BSA in PBST in a humidified chamber for 1 hr at room temperature or overnight at 4°C.

Decant the solution and wash the cells three times in PBS, 5 min each wash.

Incubate cells with the secondary antibody in 1% BSA for 1 hr at room temperature in the dark.

Decant the secondary antibody solution and wash three times with PBS for 5 min each in the dark.

​Multicolor staining (optional step)

In order to be able to examine the co-distribution of two (or more) different antigens in the same sample, a double immunofluorescence procedure can be carried out. This can be performed either simultaneously (in a mixture) or sequentially (one antigen after another).

Please ensure you have antibodies for different species and corresponding secondary antibodies. For example, rabbit antibody against antigen A, mouse antibody against antigen B. Alternatively you can use directly conjugated primary antibodies conjugated to different fluorphores.

​Simultaneous incubation

Incubate cells with 1% BSA in PBST for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 10% serum from the species that the secondary antibody was raised in).

Incubate cells in the mixture of two primary antibodies (e.g. rabbit against human target-1 and mouse against human target-2, if the targets are human proteins) in 1% BSA in PBST in a humidified chamber for 1 hr at room temperature or overnight at 4°C.

Decant the mixture solution and wash the cells three times in PBS, 5 min each wash.

Incubate cells with the mixture of two secondary antibodies which are raised in different species (with two different fluorochromes, i.e. Texas Red-conjugated against rabbit and FITC-conjugated against mouse) in 1% BSA for 1 hr at room temperature in the dark.

Decant the mixture of the secondary antibody solution and wash three times with PBS for 5 min each in the dark.

​Sequential incubation

First blocking step: incubate cells with the first serum (10% serum from the species that the secondary antibody was raised in) for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 1% BSA) at room temperature.​

Incubate cells with the first primary antibody in 1% BSA or 1% serum in PBST in a humidified chamber for 1 hr at room temperature or overnight at 4°C depending on the concentration of the antibody and the accessibility of the antigen.​

Decant the first primary antibody solution and wash the cells three times in PBS, 5 min each wash.​

Incubate cells with first secondary antibody (labelled with Fluorochrome-1) in 1% BSA in PBST for 1 hr at room temperature in the dark.​

Decant the first secondary antibody solution and wash three times with PBS for 5 min each in the dark.​

Second blocking step: incubate cells with the second serum (10% serum from the species that the secondary antibody was raised in) for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 1% BSA) at room temperature in the dark.​

Incubate cells with the second primary antibody in 1% BSA in PBST in a humidified chamber in the dark for 1 hr at room temperature or overnight at 4°C depending on the concentration of the antibody and the accessibility of the antigen.​

Decant the second primary antibody solution and wash the cells three times in PBS, 5 min each wash in the dark.​

Incubate cells with second secondary antibody (labelled with Fluorochrome-2) in 1% BSA for 1 hr at room temperature in the dark.​

Decant the second secondary antibody solution and wash three times with PBS for 5 min each in the dark.​

If you have to detect more than two antigens, continue following steps 1-5 for the rest of the antibodies.

Counter staining

Incubate cells on 0.1-1 μg/ml Hoechst or DAPI (DNA stain) for 1 min.

Rinse with PBS.

Mounting

Mount coverslip with a drop of mounting medium.

Seal coverslip with nail polish to prevent drying and movement under microscope.